Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Recombinant virus encoding one or more heterologous proteins...
Reexamination Certificate
1999-04-26
2001-04-17
Mosher, Mary E. (Department: 1648)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Recombinant virus encoding one or more heterologous proteins...
C435S235100, C435S320100, C424S093200, C424S229100, C424S232100
Reexamination Certificate
active
06217882
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to the field of recombinantly-produced proteins and vector systems, and specifically to novel, recombinant Swinepox sequences useful in vector molecules capable of expressing heterologous genes in transformed hosts and in making specialized vectors.
BACKGROUND OF THE INVENTION
Various attempts have been made to construct vaccine vectors from viruses. The use of viruses and virus proteins in eukaryotic host-vector systems has been the subject of a considerable amount of investigation and speculation. Many existing viral vector systems suffer from significant disadvantages and limitations which diminish their utility. For example, a number of eukaryotic viral vectors are either tumorigenic or oncogenic in mammalian systems, creating the potential for serious health and safety problems associated with resultant gene products and accidental infections. Further, in some eukaryotic host-viral vector systems, the gene product itself exhibits antiviral activity, thereby decreasing the yield of that protein.
In the case of simple viruses, the amount of exogenous DNA which can be packaged into a simple virus is limited. This limitation becomes a particularly acute problem when the genes used are eukaryotic. Because eukaryotic genes usually contain intervening sequences, they are too large to fit into simple viruses. In the case of complex viruses, size of exogenous DNA to be inserted is not a limiting factor. However, because they have many restriction sites, it is more difficult to insert exogenous DNA into complex viruses at specific locations.
Studies with vaccinia virus have demonstrated that poxviruses in general have several advantageous features as vaccine vectors. Poxviruses are taxonomically classified into the family Chordopoxvirinae, whose members infect vertebrate hosts, e.g., the Orthopoxvirus vaccinia. Vaccinia virus has recently been developed as a eukaryotic cloning and expression vector (Mackett, M. et al., [1985
] DNA Cloning
, Vol. II, ed. D. M. Glover, pp. 191-212, Oxford: IRL Press; Panicali, D. et al. [1982
] Proc. Natl. Acad. Sci. USA
88:5364-5368). Numerous viral antigens have been expressed using vaccinia virus vectors (Paoletti, E. et al. [1986
] Proc. Natl. Acad. Sci. USA
81:193-197; Piccine, A. et al. [1986
] BioEssays
5:248-252) including, among others, HBsAg, rabies G protein and the gp120/gp41 of human immunodeficiency virus (HIV). Regulatory sequences from the spruce budworm EPV have been used previously with vaccinia (Yuen, L. et al. [1990
] Virology
175:427-433).
The advantages of poxviruses as vaccine vectors include the ability of poxvirus-based vaccines to stimulate both cell-mediated and humoral immunity, minimal cost to mass produce vaccine and the stability of the lyophilized vaccine without refrigeration, ease of administration under non-sterile conditions, and the ability to insert at least 25,000 base pairs of foreign DNA into an infectious recombinant, thereby permitting the simultaneous expression of many antigens from one recombinant.
However, although recombinant vaccinia viruses have demonstrated great potential as vaccines, vaccinia has several drawbacks that have prevented its widespread use in both human and veterinary medicine. First, vaccinia virus has a wide host range that includes man and many domestic animals. This could permit the spread of a recombinant vaccinia vaccine to other animal populations not intended for vaccination. Secondly, although the vaccinia virus recombinants are attenuated by inactivation of the thymidine kinase gene, this virus still has the ability to cause potentially serious generalized vaccinal infections in immunocompromised individuals (i.e., patients with AIDS).
Another poxvirus, swinepox virus, the only known member of the genus Suipoxvirus, is naturally restricted to swine and occurs widely throughout the world. Swinepox virus produces a mild, self-limiting disease in swine. (Kasza et al. [1960
] Am. J. Vet. Res.
21:269-272; Shope [1940
] Arch. Gesamte. Virustorsch
1:457-467). This virus is characterized by a genome 175 kb in size which includes a thymidine kinase (TK) gene closely resembling the TK genes of other poxviruses (Feller et al. [1991
] Virol.
183:578-585).
Pseudorabies is one of the most important diseases affecting the swine industries of the USA and several countries within Europe. Losses due to disease in the USA each year run into several million dollars. This disease is characterized in its reservoir host, swine, by central nervous system disorders in suckling pigs, respiratory disease in growing pigs, and fever and inappetence in adult swine. Cattle, dogs, cats, and other species are atypical hosts of pseudorabies, but develop an invariably fatal neurological disease similar to rabies. Highly pathogenic strains of pseudorabies have been detected in the USA and later in Europe since the 1960's (Gustafson, D. P. [1986] “Pseudorabies,” in
Diseases of Swine
, pp. 274-289, 6th Edition, eds. Leman, A. D., Straw, B., Glock, R. D., Mengeling, W. I., Penny, R. H. C., and Scholl, E., Publ. ISU press, Ames, Iowa).
The control and the eradication of pseudorabies in the USA has proven to be difficult due to the existence of a large population of feral swine in the southern states which is known to be infected with pseudorabies virus.
There remains a need for a safer and effective vector system to create vaccines directed to diseases of humans and animals, including e.g., pseudorabies infections of swine.
BRIEF SUMMARY OF THE INVENTION
In one aspect, the present invention provides a recombinant swinepox virus vector which contains a heterologous gene or genes encoding a selected pathogenic immunogenic or antigenic protein under the control of a suitable regulatory sequence capable of directing the expression of said protein in a selected host cell. Preferably, the heterologous gene is inserted into the swinepox virus vector, either replacing or disrupting a naturally-occurring swinepox virus sequence or gene which is not essential to the replication of the recombinant swinepox virus vector in a selected host cell.
In one embodiment of such a vector, the heterologous gene is inserted into the thymidine kinase (TK) gene region of swinepox virus. TK is one of the proteins [SEQ ID NO: 58] encoded by a nucleic acid sequence [SEQ ID NO: 57] of the HindIII H fragment or a portion thereof, of the swinepox virus. In an illustrated embodiment, the foreign gene is a pseudorabies gene, most preferably the gp50 and/or gp63 gene. Additionally, other pseudorabies and non-pseudorabies genes are expected to be useful.
In a further aspect, the present invention provides the DNA sequences of the sense strand [SEQ ID NO: 1] and the anti-sense strand [SEQ ID NO: 14] of the approximately 14 kb HindIII C fragment of the swinepox virus. This DNA sequence contains fragments which encode about 27 swinepox virus proteins.
In a related aspect, the present invention provides the DNA sequences [SEQ ID NOS: 8-13 and 36-56], and putative amino acid sequences [SEQ ID NOS: 2-7 and 15-35] of the 27 proteins encoded by fragments from the 14 kb HindIII C fragment. Certain of these sequences from the fragment, like the TK gene, are expected to be non-essential and thus useful as locations for the insertion of, or replacement by, foreign genes for expression thereof.
In another aspect, the present invention provides the DNA sequence [SEQ ID NO: 57] of the SPV TK gene and the putative amino acid sequence [SEQ ID NO: 58] of TK.
In yet another aspect, the present invention provides a plasmid, p19SPB1, containing the full length SPV TK gene [SEQ ID NO: 57].
In a still further aspect, the present invention provides a therapeutic composition useful in treating a selected disease, which composition contains a swinepox virus vector capable of expressing a heterologous protein capable of alleviatin
Gibbs E. P. J.
Moyer Richard W.
Vinuela Eladio
Mosher Mary E.
Saliwanchik Lloyd & Saliwanchik
University of Florida Research Foundation Inc.
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